Chromatography is a known method of analyzing a sample comprised of several components to qualitatively determine the identity of the sample components as well as quantitatively determine the concentration of the components.
A typical gas chromatographic apparatus includes an injection port into which the sample is injected and mixed with an inert gas at high temperature, a column through which the various dissolved components of the sample will travel at a rate related to the characteristics of the specific components, and a detector for measuring the retention time of each component. The time between the injection of a sample and the detection of a specific component is called the retention time for that component. The results of a chromatographic separation are displayed as a plot of detector signal versus time, commonly known in the art as a chromatogram. A chromatogram typically comprises a plurality of peaks wherein each peak corresponds to a certain component of the analyzed sample. The area of the peak is to some degree characteristic of the amount of the respective component present in the sample. In order to insure a reliable qualitative and quantitative analysis of the sample, it is necessary that the chromatograph perform proper identification of those peaks in the chromatogram that represent certain compounds present in the sample.
As various components will have different retention times, the chromatogram will usually provide a series of Gaussian-shaped sample peaks wherein each peak represents a respective component in the sample. Ideally, a chromatogram of a sample containing, e.g., a plurality of components, should have a respective plurality of clearly separate and identifiable peaks. Such a chromatogram may thus be analyzed to determine the identity of the respective components by noting the time occurrence of each sample peak and comparing the observed retention time of a sample peak in the chromatogram to a characteristic retention time for a known, or standard, peak that is derived from a standard mixture of known components. Since the retention time is a unique physical characteristic of each different component, the observed retention time at which each sample peak occurs may be compared to the characteristic retention times of compounds in a standard mixture so as to allow one to assign, or name, an identity to each component in the sample under investigation.
Retention time is typically considered to be a function of the flow rate of the fluid, which in turn is a function of operating parameters such as the column dimensions or temperature, the inlet outlet pressure, and the gas viscosity. Because the retention time of a component is subject to variations in the operating parameters, retention time stability in a chromatographic system is a desirable characteristic that determines the ability of the system to properly identify closely eluting components and to allow a component to be identified within a desired identification window of time. However, retention time stability is not often achieved without use of extensive methods and apparatus for calibration. Conventional analytical methods have heretofore included techniques such as frequent recalibration so as to correct for any systematic errors or shift due to instability in the retention time.
Some conventional chromatographic equipment incorporate a form of a peak identification algorithm for improving the process of assigning compound names to the observed chromatographic peaks based on the observed retention times. These algorithms are based upon the use of a fixed retention time window for identification of a given peak. The conventional algorithm may be understood as follows. If the observed retention time of a given peak in a sample falls within a window centered on a characteristic retention time known to be associated with a compound, the respective compound name is assigned to that peak. If a plurality of peaks occur within the window, the peak nearest the known retention time for the compound is assigned the compound name.
The window size is sometimes determined as a percentage of the characteristic retention time for the given compound. However, because the retention times of components will change, a wide retention time window is often selected to capture and thus locate a given peak of interest. This step also requires intervention by the user, e.g., by editing the characteristic retention time for a given compound. Further, as the retention time window becomes larger, unwanted peaks are captured by the window and accordingly it is difficult to resolve which of the peaks within a window is the peak of interest. If the window is made too small, the system may not be able to recalibrate; and the performance of the system is then subject to drift. Some complex algorithms have been employed that track and adjust for drift between calibrations; however, this approach is nonetheless disadvantageous because each copy of an chromatographic method may be the result of a different data reduction method and the characteristic retention time for a given compound will vary according to the instrumentation used to determine the characteristic retention time. Also, the data reduction method will change when a column is replaced or reduced in length.
An improved approach was used in a microbial identification system commercially available in the Hewlett-Packard Model HP5898A Chromatograph, wherein peaks were located in an analysis of a calibration sample in a calibration sequence so that the retention times of the calibration sample peaks could be used to determine a calibration table of retention time indices. The retention time indices were then used in performing peak identification. The peak location algorithm employed an algorithm that included move, stretch, and distort parameters for improved peak location in the presence of spurious peaks due to noise and other artifacts. However, the peak location algorithm typically required that each and every peak of interest in the calibration sample be identified for the algorithm to succeed. If not, the algorithm would fail and a failure mode would be declared.
Several shortcomings remain in the above-described approaches. Firstly, it is undesirable to require that all peaks in a chromatogram must be identified for a peak identification method to succeed. Secondly, the aforementioned approaches are not amenable to the identification of groups of peaks that may have been recorded in a chromatogram generated by the use of multiple columns or according to techniques known in the art as heart cutting or multidimensional chromatography.